Recent advances in stem cell biology may make possible new approaches to the treatment of Parkinson's disease (PD), which results from a progressive degeneration and loss of dopamine producing neurons in the substantia nigra. These approaches could involve transplantation of dopaminergic (DA) neurons to replace lost cells and/or drug treatment to stimulate the body's own regenerative capabilities by promoting survival, migration/homing, proliferation, and differentiation of endogenous neuronal cells. However, such approaches will require identification of renewable cell sources of engraftable functional DA neurons, an improved ability to manipulate their proliferation and differentiation, as well as a better understanding of the signaling pathways that control neural fate and brain development. Here we propose to develop and implement high throughput cellular screen of our 100,000-membered combinatorial chemical library to identify small molecules that can selectively induce differentiation of mouse embryonic stem cells (ESC) to functional DA neurons. We will further characterize their mechanism of action (MOA) by integrated chemical and functional genomic approaches, including detailed structure-activity-relationship (SAR) studies, affinity chromatography, genome-wide mRNA expression analysis, chemical and genetic epistasis, arrayed genome-wide cDNA and siRNA complementation, and various other structural, biochemical and cellular studies. Collectively, the studies described in this proposal will generate chemical tools for producing unlimited amount of functional DA neurons for various applications, broaden our understanding of signaling pathways controlling neuronal specification, and may ultimately facilitate development of small molecule therapeutics to treat PD and stimulate in vivo regeneration of DA neurons.